This application is a national stage application filed under 35 U.S.C. xc2xa7371 of PCT International Application No. PCT/EP99/06036, filed on Aug. 18, 1999 and which claims priority from German Application No. 198 37 387.2, filed Aug. 18, 1998.
The present invention relates to a 3xe2x80x2-deoxypentopyranosylnucleic acid consisting essentially of 3xe2x80x2-deoxypentopyranosylnucleosides of the formula (I) or of the formula (II) 
their preparation and use for the production of a therapeutic, diagnostic and/or electronic component.
Pyranosylnucleic acids (p-NAs) are structural types which in general are isomeric to the natural RNA and in which the pentose units are present in the pyranose form and are repetitively linked between positions C-2xe2x80x2 and C-4xe2x80x2 by phosphodiester groups. xe2x80x9cNucleobasexe2x80x9d is understood here as meaning the canonical nucleobases A, T, U, C, G, but also the pairs isoguanine/isocytosine and 2,6-diaminopurinelxanthine and, within the meaning of the present invention, also other purines and pyrimidines. p-NAs, to be precise the p-RNAs derived from ribose, were described for the first time by Eschenmoser et al. (Helv. Chim. Acta 1993, 76, 2161; Helv. Chim Acta 1995, 78, 1621; Angew. Chem. 1996, 108, 1619-1623). They exclusively form so-called Watson-Crick-paired, i.e. purine-pyrimidine- and purine-purine-paired, antiparallel, reversibly xe2x80x9cmeltingxe2x80x9d, quasi-linear and stable duplices. Homochiral p-RNA strands of opposite chiral sense likewise pair controllably and are strictly non-helical in the duplex formed. This specificity, which is valuable for the synthesis of supramolecular units, is associated with the relatively low flexibility of the ribopyranose phosphate backbone and with the high inclination of the base plane to the strand axis and the tendency resulting from this for intercatenary base stacking in the resulting duplex and can finally be attributed to the participation of a 2xe2x80x2,4xe2x80x2-cis-disubstituted ribopyranose ring in the synthesis of the backbone. These essentially better pairing properties make p-NAs preferred pairing systems compared with DNA and RNA for application in the synthesis of supramolecular units. They form a pairing system which is orthogonal to natural nucleic acids, i.e. they do not pair with the DNAs and RNAs occurring in the natural form, which is particularly of importance in the diagnostic field.
p-RNA, however, has the following disadvantages which are to be attributed to the presence of the 3xe2x80x2-hydroxyl function:
1. The necessary protection of the 3xe2x80x2-hydroxyl group with a benzoyl protective group complicates and prolongs the synthetic route to the monomeric units considerably.
2. On account of the use of the allyl radical as a base and phosphate protective group, the deprotection and the removal of the oligonucleotide must be carried out by two successively connected steps. First, the allyl radicals are removed by the Noyori method (R. Noyori, J. Am. Chem. Soc. 1990, 112, 1691-6). Then the base-labile acyl groups must be cleaved and the oligonucleotide removed from the carrier.
3. After oligonucleotide synthesis is complete, the cleavage of the 3xe2x80x2-benzoyl radicals from the oligonucleotide causes difficulties. In order to remove these radicals effectively the use of hydrazine is necessary, which can lead to ring-opening of the pyrimidine bases, especially uracil and thymine.
4. In the synthesis of the oligonucleotides, 5-(4-nitrophenyl)-1H-tetrazole is employed as a coupling reagent in the automated p-RNA synthesis. The concentration of this reagent in the solution of tetrazole in acetonitrile is so high here that in general the 5-(4-nitrophenyl)-1H-tetrazole crystallizes out in the thin tubing of the synthesizer and the synthesis thus comes to a premature end. Moreover, it was observed that the oligomers were contaminated with 5-(4-nitrophenyl)-1H-tetrazole. The benzimidazolium triflate alternatively used also has negative points: it crystallizes out, even though rarely, in the tubing, is expensive and must moreover be recrystallized before its use.
The object of the present invention was therefore to provide and to oligomerize novel pentopyranosylnucleosides for orthogonal pairing systems, whereby the disadvantages described above can be circumvented.
Surprisingly, it has now been found that 3xe2x80x2-deoxypentopyranosylnucleic acids (p-DNA) do not have the disadvantages described and still have the advantageous orthogonal pairing properties (see FIG. 3).
A subject of the present invention is therefore a 3xe2x80x2-deoxypentopyranosylnucleic acid consisting essentially of 3xe2x80x2-deoxypentopyranosylnucleosides of the formula (I), 
in which
R1 is equal to H, OH, Hal where Hal is equal to Br or Cl, a radical selected from 
or xe2x80x94Oxe2x80x94P[N(i-Pr)2] (OCH2CH2CN) where i-Pr is equal to isopropyl, or xe2x80x94Oxe2x80x94PHxe2x80x94(xe2x95x90O)(xe2x80x94O),
R2, R3 and R4 independently of one another, identically or differently, are in each case H, NR5R6, OR7, SR8, xe2x95x90O, CnH2n+1 where n is an integer from 1-12, preferably 1-8, in particular 1-4, a xcex2-eliminable group, preferably a group of the formula xe2x80x94OCH2CH2R18 where R18 is equal to a cyano or p-nitrophenyl radical or a fluorenylmethyloxycarbonyl (Fmoc) radical, or (CnH2n)NR10R11 where R10R11 is equal to H, CnH2n+1 or R10R11 is bonded via a radical of the formula 
in which R12, R13, R14 and R15 independently of one another, identically or differently, are in each case H, OR7, where R7 has the abovementioned meaning, or CnH2+1, or CnH2xe2x88x921, where n has the abovementioned meaning, and
R5, R6, R7 and R8 independently of one another, identically or differently, are in each case H, CnH2n+1, or CnH2nxe2x88x921, where n has the above-mentioned meaning, xe2x80x94C(O)R9 where R9 is equal to a linear or branched, optionally substituted alkyl or aryl radical, preferably a phenyl radical, X, Y, and Z independently of one another, identically or differently, are in each case xe2x95x90N-, xe2x95x90C(R16)- or xe2x80x94N(R17)- where R16 and R17 independently of one another, identically or differently, are in each case H or CnH2n+1 or (CnH2n)NR10R11 with the abovementioned meanings, the dotted lines represent optional unsaturation, and
Sc1 is hydrogen or a protective group selected from an acyl, trityl, allyloxycarbonyl, a photolabile or xcex2-eliminable protective group, preferably a fluorenymethyloxycarbonyl (Fmoc) or 4, 4xe2x80x2-dimethoxytrityl (DMT) group,
or of the formula (II) 
in which R1xe2x80x2 is equal to H, OH, Hal where Hal is equal to Br or Cl, a radical selected from 
or xe2x80x94Oxe2x80x94P[N(i-Pr)2] (OCH2CH2CN) where i-Pr is equal to isopropyl, or xe2x80x94Oxe2x80x94PHxe2x80x94(xe2x95x90O)(xe2x80x94O)
R2xe2x80x2, R3xe2x80x2, and R4xe2x80x2 independently of one another, identically or differently, are in each case H, xe2x95x90O, CnH2n+1 or OCnH2nxe2x88x921, a xcex2-eliminable group, preferably a group of the formula xe2x80x94OCH2CH2R18 where R18 is equal to a cyano or p-nitrophenyl radical or a fluorenylmethyloxycarbonyl (Fmoc) radical or (CnH2n)NR10xe2x80x2R11xe2x80x2, where R10xe2x80x2, R11xe2x80x2, independently of one another has the abovementioned meaning of R10 or R11, and
Xxe2x80x2, Yxe2x80x2, and Zxe2x80x2 independently of one another, identically or differently, are in each case xe2x95x90Nxe2x80x94, xe2x95x90C(R16xe2x80x2)xe2x80x94 or xe2x80x94N(R17xe2x80x2), where R16xe2x80x2and R17xe2x80x2independently of one another have the abovementioned meaning of R16 and R17, the dotted lines represent optional unsaturation, and Sc1xe2x80x2, has the abovementioned meaning of Sc1.
According to the present invention, the nucleic acid according to the invention is synthesized from 3xe2x80x2-deoxypentopyranosylnucleosides, further modifications, such as the conjugates described in greater detail below, likewise being included by the invention.
The 3xe2x80x2-deoxypentopyranosylnucleosides are in general 3xe2x80x2-deoxyribo-, 3xe2x80x2-deoxyarabino-, 3xe2x80x2-deoxylyxo- and/or 3xe2x80x2-deoxyxylopyranosylnucleosides, preferably 3xe2x80x2-deoxyribopyranosyl-nucleosides, where the 3xe2x80x2-deoxypentopyranosyl part can be of D configuration, but also of L configuration.
Customarily, the 3xe2x80x2-deoxypentopyranosylnucleosides are 3xe2x80x2-deoxypentopyranosylpurine, -2,6-diaminopurine, -6-purinethiol, -pyridine, -pyrimidine, -adenosine, -guanosine, -isoguanosine, -6-thioguanosine, -xanthine, -hypoxanthine, -thymidine, -cytosine, -isocytosine, -indole, -tryptamine, -N-phthaloyltryptamine, -uracil, -caffeine, -theobromine, -theophylline, -benzotriazole or -acridine, in particular 3xe2x80x2-deoxypentopyranosylpurine, -pyrimidine, -adenosine, -guanosine, -thymidine, -cytosine, tryptamine, -N-phthalotryptamine or -uracil.
The compounds include 3xe2x80x2-deoxypentopyranosylnucleosides which can be used as linkers, i.e. as compounds having functional groups which can bind covalently to biomolecules, such as, nucleic acids occurring in their natural form or modified nucleic acids, such as DNA, RNA but also p-NAs, preferably p-DNAs.
For example, among these are included 3xe2x80x2-deoxypentopyranosylnucleosides in which R2, R3, R4, R2xe2x80x2, R3xe2x80x2 and/or R4xe2x80x2 is a 2-phthalimidoethyl or allyloxy radical. Preferred linkers according to the present invention are, for example, uracil-based linkers in which the 5-position of the uracil has preferably been modified, e.g. N-phthaloylaminoethyluracil, but also indole-based linkers, preferably tryptamine derivatives, such as N-phthaloyltryptamine.
Moreover, the present invention includes 3xe2x80x2-deoxypentopyranosylnucleosides which carry a protective group, preferably an acid-, base-, photolabile- or xcex2-eliminable protective group, in particular a trityl group, particularly preferably a dimethoxytrityl group, exclusively on the 4xe2x80x2-oxygen atom of the 3xe2x80x2-deoxypentopyranoside moiety.
The following compounds are preferred examples of pentopyranosylnucleosides which can be present in the nucleic acid according to the invention, or are particularly suitable for its preparation:
A) [(2xe2x80x2,4xe2x80x2-Di-O-benzoyl)-3xe2x80x2-deoxy-xcex2-ribopyranosyl]nucleosides, in particular a [(2xe2x80x2,4xe2x80x2-di-O-benzoyl)-3xe2x80x2-deoxy-xcex2-ribopyranosyl]adenine, -guanine, -cytosine, -thymidine, -uracil, -xanthine or -hypoxanthine, and also an N-benzoyl-2xe2x80x2,4xe2x80x2-di-O-benzoyl-3xe2x80x2-deoxyribopyranosylnucleoside, in particular an -adenine, -guanine or -cytosine, and also an N-isobutyroyl-2xe2x80x2,4xe2x80x2-di-O-benzyl-3xe2x80x2-deoxyribopyranosylnucleoside, in particular an -adenine, -guanine or -cytosine, and also an O6-(2-cyanoethyl)-N2-isobutyroyl-2xe2x80x2,4xe2x80x2-di-O-benzoyl-3xe2x80x2-deoxyribopyranosylnucleoside, in particular a -guanine, and also an O6-(2-(4-nitrophenyl)ethyl)-N2-isobutyroyl-2xe2x80x2,4xe2x80x2-di-O-benzoyl-3xe2x80x2-deoxyribopyranosylnucleoside, in particular a -guanine.
B) 3xe2x80x2-Deoxy-xcex2-ribopyranosylnucleosides, in particular a 3xe2x80x2-deoxy-xcex2-ribopyranosyladenine, -guanine, -cytosine, -thymidine, -uracil, -xanthine or hypoxanthine, and an N-benzoyl-, N-isobutyroyl-, O6-(2-cyanoethyl)- or O6-(2-(4-nitrophenyl)ethyl)-N2-isobutyroyl-3xe2x80x2-deoxy-xcex2-ribopyranosylnucleoside, in particular a -guanine.
C) 4xe2x80x2-DMT-3xe2x80x2-deoxypentopyranosylnucleosides, preferably a 4xe2x80x2-DMT-3xe2x80x2-deoxy-ribopyranosylnucleoside, in particular a 4xe2x80x2-DMT-3xe2x80x2-deoxyribopyranosyladenine, -guanine, -cytosine, -thymidine, -uracil, -xanthine or -hypoxanthine, and also an N-benzoyl-4xe2x80x2-DMT-3xe2x80x2-deoxyribopyranosylnucleoside, in particular N-benzoyl-4xe2x80x2-DMT-3xe2x80x2-deoxyribopyranosyl-adenine, -guanine or -cytosine, and also an N-isobutyroyl-4xe2x80x2-DMT-3xe2x80x2-deoxyribopyranosylnucleoside, in particular an N-isobutyroyl-4xe2x80x2-DMT-3xe2x80x2-deoxyribopyranosyladenine, -guanine or -cytosine and also an O6-(2-cyanoethyl)-N2-isobutyroyl-4xe2x80x2-DMT-3xe2x80x2-deoxyribopyranosylnucleoside, in particular an O6-(2-cyanoethyl)-N2-isobutyroyl-4xe2x80x2-DMT-3xe2x80x2-deoxyribopyranosylguanine, and also an O6-(2-(4-nitrophenyl)ethyl)-N2-isobutyroyl-4xe2x80x2-DMT-3xe2x80x2-deoxyribopyranosylnucleoside, in particular an O6-(2-(4-nitrophenyl)ethyl)-N2-isobutyroyl-4xe2x80x2-DMT-3xe2x80x2-deoxyribopyranosylguanine.
D) 3xe2x80x2-Deoxy-xcex2-ribopyranosyl-N,Nxe2x80x2-dibenzoyladenosine or 3xe2x80x2-deoxy-xcex2-ribopyranosyl-N,Nxe2x80x2-dibenzoylguanosine.
Suitable precursors for the oligonucleotide synthesis are, for example, 4xe2x80x2-DMT-3xe2x80x2-deoxypentopyranosylnucleosides-2xe2x80x2-phosphitamide/-H-phosphonate, preferably a 4xe2x80x2-DMT-3xe2x80x2-deoxyribopyranosylnucleoside-2xe2x80x2-phosphitamide/-H-phosphonate, in particular 4xe2x80x2-DMT-3xe2x80x2-deoxyribopyranosyladenine-, -guanine-, -cytosine-, -thymidine-, -xanthine-, -hypoxanthine-, or -uracil-2xe2x80x2-phosphitamide/-H-phosphonate, and also an N-benzoyl-4xe2x80x2-DMT-3xe2x80x2-deoxyribopyranosyladenine-, -guanine- or -cytosine-2xe2x80x2-phosphitamide/-H-phosphonate and also an N-isobutyroyl-4xe2x80x2-DMT-3xe2x80x2-deoxyribopyranosyladenine-, -guanine- or -cytosine-2xe2x80x2-phosphitamide/-H-phosphonate, O6-(2-cyanoethyl)-4xe2x80x2-DMT-3xe2x80x2-deoxyribopyranosylguanine-, -xanthine-, -hypoxanthine-2xe2x80x2-phosphitamide/-H-phosphonate or O6-(2-(4-nitrophenyl)ethyl)-N2-isobutyroyl-4xe2x80x2-DMT-3xe2x80x2-deoxyribopyranosylguanine, and for the coupling to the solid carrier, for example, 4xe2x80x2-DMT-3xe2x80x2-deoxypentopyranosylnucleosides-2xe2x80x2-succinate, preferably a 4xe2x80x2-DMT-3xe2x80x2-deoxyribopyranosylnucleoside-2xe2x80x2-succinate, in particular 4xe2x80x2-DMT-3xe2x80x2-deoxyribopyranosyladenine-, -guanine-, -cytosine-, -thymidine-, -xanthine-, -hypoxanthine- or -uracil-2xe2x80x2-succinate and also an N-benzoyl-4xe2x80x2-DMT-3xe2x80x2-deoxyribopyranosyladenine-, -guanine- or -cytosine-2xe2x80x2-succinate and also an N-isobutyroyl-4xe2x80x2-DMT-3xe2x80x2-deoxyribopyranosyladenine-, -guanine- or -cytosine-2xe2x80x2-succinate, O-(2-cyanoethyl)-4xe2x80x2-DMT-3xe2x80x2-deoxyribopyranosylguanine-2xe2x80x2-succinate and also an O6-(2-(4-nitrophenyl)ethyl)-N2-isobutyroyl-4xe2x80x2-DMT-3xe2x80x2-deoxyribopyranosylguanine-2xe2x80x2-succinate.
The 3xe2x80x2-deoxyribopyranosylnucleosides can be prepared, for example, by a process in which
(a) an optionally protected nucleobase is reacted with a protected 3xe2x80x2-deoxyribopyranose,
(b) the protective groups are cleaved from the 3xe2x80x2-deoxyribopyranosyl moiety of the product from step (a) and, if appropriate,
(c) the product from step (b) is protected in the 4xe2x80x2-position of the 3xe2x80x2-deoxypentopyranoside.
In a particular embodiment, the 3xe2x80x2-deoxypyranosylnucleoside is protected by a protective group Sc1, or Sc1xe2x80x2 which is acid-labile, base-labile, photolabile, xcex2-eliminable or cleavable by metal catalysis.
In general, the protective groups mentioned are a xcex2-eliminable protective group, preferably a fluorenylmethyloxycarbonyl (Fmoc) group, a photolabile group, an acyl group, preferably an acetyl, benzoyl, nitrobenzoyl and/or methoxybenzoyl group, or trityl groups, preferably a 4,4xe2x80x2-dimethoxytrityl (DMT) group.
The introduction of a DMT group is thus carried out, for example, by reaction with DMTCl in the presence of a base, e.g. of N-ethyldiisopropylamine (Hxc3xcnig base), and, for example, of pyridine, methylene chloride or a pyridine/methylene chloride mixture at room temperature.
In general, the preferably anomerically pure ribopyranosyl structural unit is prepared starting from 1,2-O-isopropylidene-5-O-triphenylmethyl-xcex1-D-xyloftiranose (1 in FIG. 1) according to known processes (W. Sowa, Can. J. Chem, 1968, 46, 1568; Z. J. Witczak et. al. Carbohydrate Research, 1982, 110. 326) but in general with improved yields. Analogously to the known process, 1 (FIG. 1) is tritylated and sodium hydride is preferably used instead of sodium hydroxide solution for the preparation of the methyl xanthate ester in the 3xe2x80x2-position (2 in FIG. 1). After removal of the methyl xanthate, the trityl and the isopropylidene protective groups are preferably cleaved using trifluoroacetic acid instead of the 80% glacial acetic acid described in the literature. The yields were in some cases improved considerably by means of these modifications.
In a further embodiment, a linker according to formula (II), in which R4xe2x80x2 is (CnH2n)NR10xe2x80x2R11xe2x80x2 and R10xe2x80x2R11xe2x80x2 is linked via a radical of the formula (III) having the meaning already designated, is advantageously prepared by the following process:
(a) a compound of the formula (II) where R4xe2x80x2 is equal to (CnH2n)OSc3 or (CnH2n)Hal, in which n has the abovementioned meaning, Sc3 is a protective group, preferably a mesylate group, and Hal is chlorine or bromine, is reacted with an azide, preferably in DMF, then
(b) the reaction product from (a), is preferably reduced with triphenylphosphine for example in pyridine, then
(c) the reaction product from (b) is reacted with an appropriate phthalimide, e.g. N-ethoxycarbonylphthalimide, and
(d) the reaction product from (c) is reacted with an appropriate protected pyranose, e.g. 2xe2x80x2,4xe2x80x2-di-O-benzoyl-3xe2x80x2-deoxyribopyranose, and finally
(e) the protective groups are cleaved, e.g. with methylate, and
(f) the further steps are carried out as already described above.
In addition, indole derivatives as linkers have the advantage of the ability to fluoresce and are therefore particularly preferred for nanotechnology applications in which it may be a matter of detecting very small amounts of substance. Thus, indole-1-ribosides have already been described in N. N. Suvorov et al., Biol. Aktivn. Soedin., Akad. Nauk SSSR 1965, 60 and Tetrahedron 1967, 23, 4653. However, there is no analogous process for preparing 3-substituted derivatives. In general, their preparation takes place via the formation of an aminal of the unprotected sugar component and an indoline, which is then converted into the indole-1-riboside by oxidation. For example, indole-1-glucosides and -1-arabinosides have been described (Y. V. Dobriynin et al, Khim.-Farm. Zh. 1978, 12, 33), whose 3-substituted derivatives were usually prepared by means of Vielsmeier reaction. This route for the introduction of aminoethyl units into the 3-position of the indole is too complicated, however, for industrial application.
In a further preferred embodiment, a linker according to formula (I), in which X and Y independently of one another, identically or differently, are in each case xe2x95x90C(R16) where R16 is equal to H or CnH2n and Z xe2x95x90C(R16)- where R16 is equal to (CnH2n)NR10R11 is therefore advantageously prepared by the following process:
(a) the appropriate indoline, e.g. N-phthaloyltryptamine, is reacted with a pyranose, e.g. D-3xe2x80x2-deoxyribose, to give the nucleoside diol, then
(b) the hydroxyl groups of the pyranosyl moiety of the product from (a) are preferably protected with acyl groups, e.g. by means of acetic anhydride, then
(c) the product from (b) is oxidized, e.g. by 2,3-dichloro-5,6-dicyanoparaquinone, and
(d) the hydroxyl protective groups of the pyranosyl moiety of the product from (c) are cleaved, for example, by means of methylate and then
(e) the further steps as already described above are carried out.
In a further embodiment, the 4xe2x80x2- or 2xe2x80x2-protected, 3xe2x80x2-deoxypentopyranosylnucleosides are phosphitylated in a further step or bonded to a solid phase.
Phosphitylation is carried out, for example, by means of cyanoethyl N-diisopropylchlorophosphoramidite in the presence of a base, e.g. N-ethyldiisopropylamine or by means of phosphorus trichloride and imidazole or tetrazole and subsequent hydrolysis with addition of base. In the first case, the product is a phosphoramidite and in the second case an H-phosphonate. The bonding of a protected pentopyranosylnucleoside according to the invention to a solid phase, e.g. long-chain alkylamino controlled pore glass (CPG, Sigma Chemie, Munich) can be carried out, for example, via a succinoyl linker.
The compounds obtained can be used for the preparation of the 3xe2x80x2-deoxypentopyranosylnucleic acids according to the invention.
A further subject of the present invention is therefore a process for the preparation of a 3xe2x80x2-deoxypentopyranosylnucleic acid, having the following steps:
(a) in a first step, a protected 3xe2x80x2-deoxypentopyranosylnucleoside, such as already described above, is bonded to a solid phase and
(b) in a second step the 4xe2x80x2-protected 3xe2x80x2-deoxypentopyranosylnucleoside bonded to a solid phase according to step (a) is lengthened by a 2xe2x80x2-phosphitylated 4xe2x80x2-protected 3xe2x80x2-deoxypentopyranosylnucleoside and, when using phosphoramidites, is then oxidized, for example, by an aqueous iodine solution, and
(c) step (b) is repeated with identical or different phosphitylated 3xe2x80x2-, 4xe2x80x2-protected 3xe2x80x2-deoxypentopyranosylnucleosides until the desired 3xe2x80x2-deoxypentopyranosylnucleic acid is present.
When using H-phosphonates, the oxidation to the corresponding phosphoric acid diesters is in general carried out at the end of the reaction chain, e.g. by an aqueous iodine solution.
A suitable coupling reagent when using phosphoramidites is particularly pyridinium hydrochloride, as in contrast to coupling reagents customarily used no recrystallization of the coupling reagent, no blockage of the coupling reagent lines and a significantly more rapid condensation takes place.
Suitable coupling reagents when using H-phosphonates are particularly arylsulphonyl chlorides, diphenyl chlorophosphate, pivaloyl chloride or adamantoyl chloride.
A significant advantage of the H-phosphonate method is that no phosphate protective groups are needed. The acyl protective groups of the bases can be cleaved, for example, by aqueous ammonia. When using the 2-(4-nitrophenyl)ethyl radical as a protective group for the O6-position of guanine, this can be removed, for example, without problems by treatment for about 40 minutes with 1M DBU.
It is furthermore advantageous that no protective group-cleaving hydrazinolysis of oligonucleotides is necessary, and thus no ring-opening is to be feared, especially in the case of uracil and thymine. The cyanoethyl radicals can be cleaved by aqueous ammonia together with the acyl protective groups of the bases. When using the 2-(4-nitrophenyl)ethyl radical as a protective group for the O6-position of guanine, the radical can be removed without problems by treatment for about 40 minutes with 1M DBU.
In a further particular embodiment, pentofuranosylnucleosides e.g. the adenosine, guanosine, cytidine, thymidine and/or uracil occurring in its natural form, can also be incorporated in step (a) and/or step (b), which leads, for example, to a mixed p-DNA/DNA or p-DNA/RNA.
p-NAs and in particular the p-DNAs form stable duplices with one another and in general do not pair with the DNAs and RNAs occurring in their natural form. This property makes p-NAs preferred pairing systems.
Such pairing systems are supramolecular systems of non-covalent interaction, which are distinguished by selectivity, stability and reversibility, and whose properties are preferably influenced thermodynamically, i.e. by temperature, pH and concentration. Such pairing systems can also be used, for example, on account of their selective properties as a xe2x80x9cmolecular adhesivexe2x80x9d for the bringing together of different metal clusters to give cluster associates having potentially novel properties [see, for example, R. L. Letsinger, et al., Nature 1996, 382, 607-9; P. G. Schultz et al., Nature 1996, 382, 609-11]. Consequently, the p-NAs are also suitable for use in the field of nanotechnology, for example for the preparation of novel materials, diagnostics and therapeutics and also microelectronic, photonic or optoelectronic components and for example the controlled bringing together of molecular species to give supramolecular units, such as for the (combinatorial) synthesis of protein assemblies [see, for example, A. Lombardi, J. W. Bryson, W. F. DeGrado, Biomolekuils (Pept. Sci.) 1997, 40, 495-504], as p-NAs form pairing systems which are strongly and thermodynamically controllable. A further application therefore arises, especially in the diagnostic and drug discovery field, due to the possibility of providing functional, preferably biological units such as proteins or DNA/RNA sections with a p-NA code which does not interfere with the natural nucleic acids (see, for example, WO93/20242).
Another subject of the invention is therefore the use of a 3xe2x80x2-deoxypentopyranosylnucleic acid according to the invention for the production of a medicament, in particular of a therapeutic, diagnostic and/or electronic component.
A biomolecule, e.g. DNA or RNA, can be used for non-covalent linking with another biomolecule, e.g. DNA or RNA, if both biomolecules contain sections which, as a result of complementary sequences of nucleobases, can bind to one another by formation of hydrogen bridges. Biomolecules of this type are used, for example, in analytical systems for signal amplification, where a DNA molecule whose sequence is to be analysed is on the one hand to be immobilized on a solid support by means of such a non-covalent DNA linker, and on the other hand is to be bonded to a signal-amplifying branched DNA molecule (bDNA) (see FIG. 3 in S. Urdea, Bio/Technol. 1994, 12, 926 or U.S. Pat. No. 5,624,802). A significant disadvantage of the last-described systems is that to date they are subject with respect to sensitivity to the processes for nucleic acid diagnosis by polymerase chain reaction (PCR) (K. Mullis, Methods Enzymol. 1987, 155, 335). This is to be attributed, inter alia, to the fact that the non-covalent bonding of the solid support to the DNA molecule to be analysed as well as the non-covalent bonding of the DNA molecule to be analysed does not always take place specifically, as a result of which a mixing of the functions xe2x80x9csequence recognitionxe2x80x9d and xe2x80x9cnon-covalent bondingxe2x80x9d occurs. The use of p-NA""s as an orthogonal pairing system which does not intervene in the DNA or RNA pairing process solves this problem advantageously, as a result of which the sensitivity of the analytical processes described can be markedly increased.
A further subject of the present invention is therefore a conjugate comprising a 3xe2x80x2-deoxypentopyranosylnucleoside of the formula (I) or (II) according to the invention and a biomolecule.
Biomolecule is understood within the meaning of the present invention as meaning a naturally occurring substance or a substance derived from a naturally occurring substance.
Conjugates within the meaning of the present invention are covalently bonded hybrids of p-NA""s and other biomolecules, preferably a peptide, protein or a nucleic acid, for example an antibody or a functional moiety thereof or a DNA and/or RNA occurring in its natural form. Functional moieties of antibodies are, for example, Fv fragments (Skerra and Plxc3xcckthun (1988) Science 240, 1038), single-chain Fv fragments (scFv; Bird et al. (1988), Science 242, 423; Huston et al. (1988) Proc. Natl. Acad. Sci. U.S.A., 85, 5879) or Fab fragments (Better et al. (1988) Science 240, 1041).
In a preferred embodiment, there are in this case p-DNA/DNA or p-DNA/RNA conjugates.
Conjugates are preferably used when the functions xe2x80x9csequence recognitionxe2x80x9d and xe2x80x9cnon-covalent bondingxe2x80x9d must be realized in a molecule, since the conjugates according to the invention contain two pairing systems which are orthogonal to one another.
Both sequential and convergent processes are suitable for the preparation of conjugates.
In a sequential process, for example, for example after automated synthesis of a p-RNA oligomer has taken place directly on the same synthesizerxe2x80x94after readjustment of the reagents and of the coupling protocolxe2x80x94a DNA oligonucleotide, for example, is additionally synthesized. This process can also be carried out in the reverse sequence.
In a convergent process, for example, p-RNA oligomers having aminoterminal linkers and, for example, DNA oligomers having, for example, thiol linkers are synthesized in separate operations. An iodoacetylation of the p-DNA oligomer and the coupling of the two units according to the protocols known from the literature (T. Zhu et al., Bioconjug. Chem. 1994, 5, 312) is then preferably carried out.
Convergent processes prove to be particularly preferred on account of their flexibility. The term conjugate within the meaning of the present invention is also understood as meaning so-called arrays. Arrays are arrangements of immobilized recognition species which, especially in analysis and diagnosis, play an important role in the simultaneous determination of analytes. Examples are peptide arrays (Fodor et al., Nature 1993, 364, 555) and nucleic acid arrays (Southern et al. Genomics 1992, 13, 1008; Heller, U.S. Pat. No. 5,632,957). A higher flexibility of these arrays can be achieved by binding the recognition species to coding oligonucleotides and the associated, complementary strands to certain positions on a solid carrier. By applying the coded recognition species to the xe2x80x9canti-codedxe2x80x9d solid carrier and adjustment of hybridization conditions, the recognition species are non-covalently bonded to the desired positions. As a result, various types of recognition species, such as DNA sections, antibodies, can only be arranged simultaneously on a solid carrier by use of hybridization conditions (see FIG. 4). As a prerequisite for this, however, codons and anticodons are necessary which are extremely strong and selectivexe2x80x94in order to keep the coding sections as short as possiblexe2x80x94and do not interfere with natural nucleic acids. p-NAs, preferably p-DNA""s, are particularly advantageously suitable for this.
The present invention therefore also relates to a process using in which recognition species, preferably natural DNA or RNA strands and proteins, in this case preferably antibodies or functional moieties of antibodies, are clearly encoded by p-NA sections, preferably p-DNA sections. These can then be hybridized with the associated codons on a solid carrier according to FIG. 4. Thus, always novel, diagnostically useful arrays can be synthesized on a solid support which is equipped with codons in the form of an array merely by adjustment of hybridization conditions using always novel combinations of recognition species in the desired positions. If the analyte, for example a biological sample such as serum or the like, is then applied, the species to be detected are then bonded to the array in a certain pattern which can then be recorded indirectly (e.g. by fluorescence labelling of the recognition species) or directly (e.g. by impedance measurement at the point of linkage to the codon). The hybridization is then ended by suitable condition (temperature, salts, solvents, electrophoretic processes) so that again only the carrier with the codons remains. This is then again loaded with other recognition species and is used, for example, for the same analyte for the determination of another pattern. The always new arrangement of recognition species in the array format and the use of p-NAs as pairing systems is particularly advantageous compared with other systems, see, for example WO 96/13522 (see 16, below).
A further subject of the present invention therefore in particular also relates to a diagnostic and/or an electronic component comprising a conjugate according to the invention, as already described in greater detail above.